Mass spectrometer and measurement system using the mass spectrometer

ABSTRACT

A practical mass spectrometer for proteome analysis is provided. In an ion trap-connected, orthogonal acceleration type time-of-flight mass spectrometer, the mass-to-charge ratio range that may be analyzed by one procedure is increased by providing means for reducing the velocity of ions ejected from an ion trap. The efficiency in protein identification in proteome analysis is thereby improved.

[0001] This application claims priority to Japanese Patent ApplicationNo. 2001-312118 filed on Oct. 10, 2001.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] The present invention relates to a time-of-flight massspectrometer with an ion trap bound thereto and, more particularly, to amass spectrometer for proteome analysis.

[0004] 2. Description of the Background

[0005] In the field of proteome analysis, the so-called “shotgun method”is in wide use, which comprises decomposing a protein mixture extractedfrom cells with a digestive enzyme, separating the fragment peptidesobtained using a liquid chromatograph, selecting, within a massspectrometer, one peptide species and decomposing this bycollision-induced dissociation (CID), determining the molecular weightsof the resulting fragments from a mass spectrum of the fragments, andidentifying the original protein by checking against a genome database.The technique comprising selecting and decomposing one ion specieswithin a mass spectrometer and subjecting the fragments to massspectrometry is generally called “MS/MS analysis.” In some kinds of massspectrometers, it is possible to select one fragment among the fragmentsresulting from MS/MS analysis and further subjecting that fragment toMS/MS. It is also possible to repeat such sequence n times, and thistechnique is generally called “MSn analysis.”

[0006] A quadrupole ion trap mass spectrometer (ITMS) can perform MSnanalysis where n is not less than 3, and is characterized in that highlevels of sensitivity and efficiency can be attained because CID isperformed after accumulation of ions in the ion trap. In proteomeanalysis, however, mass-to-charge ratio ranges of up to about 3,000 anda mass resolution of at least about 5,000 are desired, whereas theconventional ion trap mass spectrometers are generally about 2,000 inmass-to-charge ratio and in mass resolution and have a decreased massaccuracy. Hence, the range of application of conventional ITMS islimited, and only low protein identification efficiency can be securedwith such apparatuses.

[0007] In B. M. Chien, S. M. Michael and D. M. Lubman, Rapid Commun.Mass Spectrom. Vol.7 (1993) 837, there is disclosed a mass spectrometercomprising a quadrupole ion trap and a time-of-flight mass spectrometer(TOFMS) that are coaxially combined. When this apparatus is used, it ispossible to perform MSn analysis (n being not less than 3) at highlevels of mass-to-charge ratio ranges and mass accuracy using the TOFMS.

[0008] However, because, in this apparatus, the ion trap and the TOFMSare combined coaxially and the ion trap also serves as an acceleratorfor the TOFMS, a collision of ions with the neutral gas for CID occursfrequently during acceleration. The ions are thereby scattered and, as aresult, it is difficult to attain a high level of resolution. However,when the acceleration voltage is increased, it becomes possible to ejections in a shorter time and to thereby reduce the scattering thereof.Hence, the resolution may be improved, but there arises the problem thatthe collision energy increases and, as a result, ions are readilydecomposed. When ions are decomposed during acceleration, chemicalnoises are produced, whereby the lower detection limit is deteriorated.

[0009] In the mass spectrometer described in U.S. Pat. No. 6,011,259,CID is effected in a multi-pole ion guide, and the resulting ions aredischarged from the ion guide and analyzed in a TOFMS of the orthogonalaccelerator type. Because the orthogonal accelerator can be disposed ina high vacuum region, the frequency of collisions with a neutral gasduring acceleration is substantially negligible. Generally, theefficiency of CID in a multi-pole ion guide is lower as compared withion traps. However, the CID efficiency can be improved to some extent bycausing the ion guide to function as a two-dimensional ion trap (alsocalled a linear trap).

[0010] However, the space distribution and energy distribution of ionsrelative to the axial direction of the ion guide are large, and,therefore, the ions accelerated are dispersed. As a result, there arisesthe problem that the detection sensitivity is low. Unlike the quadrupoleion trap, the linear trap cannot be used in MSn where n is not less than3.

[0011] In C. Marinach, A. Brunot, C. Beaugrand, G. Bolbach, J. -C.Tabet, Proceedings of the 49^(th) ASMS Conference on Mass Spectrometryand Allied Topics, Chicago, Ill., May 27-31, 2001, there is disclosed amass spectrometer in which a quadrupole ion trap and a TOFMS arecombined off axis. In this apparatus, ions are initially ejected fromthe ion trap, then accelerated in a direction perpendicular to the axisof the ion trap, and finally subjected to analysis on the TOFMS. In thisapparatus, ions spatially focused in the middle of the ion trap aredispersed as far as possible relative to the axial direction duringtransfer thereof from the ion trap to the orthogonal accelerator. Thiscauses the ions to form a continuous ion flow while an accelerationvoltage pulse is continuously applied at spaced intervals (i.e.,repeated pulses) to perform analysis on the TOFMS. Since ions spatiallyand energetically focused within the ion trap are converted to acontinuous ion flow, there arises, as a result, the same problems aswith the apparatus described above with reference to U.S. Pat. No.6,011,259.

[0012] As discussed above, the prior art mass spectrometers arecharacterized in that it is difficult to simultaneously attain broadmass-to-charge ratio ranges and high mass resolution with sufficientdetection sensitivity.

SUMMARY OF THE INVENTION

[0013] The present invention preferably addresses the above limitationsby providing a mass spectrometer that combines an ion trap with a TOFMSof the orthogonal acceleration type. In the mass spectrometer accordingto the present invention, the ions ejected from the ion trap aretransferred to the orthogonal accelerator, and an acceleration voltageis applied thereto in the transverse direction relative to the directionof ion flow. According to the invention, the mass-to-charge ranges arecontrolled by setting the time from ion ejection from the ion trap toacceleration voltage pulse application at predetermined values.

[0014] As a means for ejecting ions from the ion trap, an acceleratingelectric field may be formed within the ion trap after stopping theapplication of an RF voltage for accumulating ions. When an acceleratingelectric field is formed under application of an RF voltage, the spatialdistribution of ions within the ion trap, the kinetic energydistribution among ions within the ion trap, and the spatialdistribution of ions in the acceleration region due to impact scatteringby collision with natural gases increase. The conventional methodsmentioned above do not produce such increasing effects.

[0015] Even when the above-mentioned means for ejecting ions isprovided, the initial voltage at which ions are ejected varies accordingto the initial location of ions. Those ions located on the remote sideof the ion trap from the outlet are ejected later than the ionsoccurring on the side closer to the outlet. Because, however, thevelocity of the former is higher than the ions occurring on the sidecloser to the outlet, the former ions pass the latter at a certainlocation. This location is called the “space focal plane.” By forming anelectric field for accelerating ions in the direction of movementthereof between the ion trap outlet and the orthogonal accelerator, itis possible to adjust the position of the space focal plane according tothe well-known principle of multi-stage acceleration. By optimizing theposition of the space focal plane according to this principle, itbecomes possible to improve the efficiency of detection of ionsoccurring in the acceleration region boundary.

[0016] Further, means may be provided for reducing the velocitydistribution of ions during transfer thereof from the ion trap to theorthogonal accelerator. The means for reducing the velocity distributionof ions may be disposed within the ion trap or outside of the same.

[0017] Ions ejected from the ion trap arrive at the orthogonalaccelerator at different times according to their mass-to-charge ratios(m/z), and only those ions that are in the acceleration region at thetime of acceleration voltage application (pulsing) are accelerated inthe orthogonal accelerator and sent to the detector. That is, the rangeof mass-to-charge ratios of ions analyzed by a single pulse in the iontrap is restricted by the length of the orthogonal accelerator and thelength of the detector, among others. Therefore, the mass-to-chargeratio range which may be analyzed at a single time is physicallylimited. Although the mass-to-charge ratio range may be broadened byincreasing the length of the orthogonal accelerator, the ion beamspreading in the acceleration region then increases, and it becomesdifficult to realize a high resolution over the entire range. It is alsonecessary to increase the size of the detector corresponding to thelength of the acceleration region. However, the detector may beexpensive, and the cost thereof largely depends on the size of thedetector.

[0018] By providing means for reducing the velocity distribution of theions entering the acceleration region, it is possible to broaden themass-to-charge ratio range analyzable by one process of ion accumulationin the ion trap. Such extension of the mass-to-charge ratio range isuseful in proteome analysis, in particular.

[0019] Specific means available for reducing the ion velocitydistribution in the axial direction include: (1) increasing theacceleration electric field during the period until ions are ejectedfrom the ion trap; or (2) varying the electric field in the region fromthe ion trap outlet to the orthogonal accelerator inlet, or in a part ofthat region after ion ejection from the ion trap.

[0020] Other means for enlarging the mass-to-charge ratio range than thereduction of the ion velocity distribution include techniquescomprising: (3) dividing the mass-to-charge ratio range to be analyzedinto a plurality of ranges, analyzing each divided region, and combiningthe data thus obtained; or (4) analyzing those ions in a lowmass-to-charge ratio range among the ions accumulated in the ion trap byion trap mass spectrometry and analyzing the remaining ions using aTOFMS of the orthogonal acceleration type. By combining the ion trap andan orthogonal acceleration type TOFMS, it is possible to further enlargethe mass-to-charge ratio range.

BRIEF DESCRIPTION OF THE DRAWINGS

[0021] For the present invention to be clearly understood and readilypracticed, the present invention will be described in conjunction withthe following figures, wherein like reference characters designate thesame or similar elements, which figures are incorporated into andconstitute a part of the specification, wherein:

[0022]FIG. 1 shows the constitution of a mass spectrometer according tothe present invention;

[0023]FIG. 2 shows the voltage sequence in a mass spectrometer accordingto the invention;

[0024]FIG. 3 shows the constitution of a plane electrode type quadrupoleion trap adequate for use in the practice of invention;

[0025]FIG. 4 shows a first method of ion trap control by which the ionvelocity distribution may be reduced;

[0026]FIG. 5 schematically shows the mass-to-charge ratio rangeincreasing effect which may be produced by reducing the ion velocitydistribution;

[0027]FIG. 6 shows a second method of ion trap control by which the ionvelocity distribution may be reduced;

[0028]FIG. 7 shows the constitution of an electrode constitution and amethod of controlling the same by which the ion velocity distributionmay be reduced;

[0029]FIG. 8 shows the results of calculation indicating themass-to-charge ratio range increasing effect;

[0030]FIG. 9 illustrates the segment method according to the invention;

[0031]FIG. 10 shows the constitution of a hybrid apparatus according tothe invention; and

[0032]FIG. 11 shows the constitution of another mass spectrometeraccording to the present invention.

DETAILED DESCRIPTION OF THE INVENTION

[0033] It is to be understood that the figures and descriptions of thepresent invention have been simplified to illustrate elements that arerelevant for a clear understanding of the present invention, whileeliminating, for purposes of clarity, other elements that may be wellknown. Those of ordinary skill in the art will recognize that otherelements are desirable and/or required in order to implement the presentinvention. However, because such elements are well known in the art, andbecause they do not facilitate a better understanding of the presentinvention, a discussion of such elements is not provided herein. Thedetailed description will be provided hereinbelow with reference to theattached drawings.

[0034] First Exemplary Embodiment

[0035]FIG. 1 shows a mass spectrometer according to the presentinvention and a measurement system using the same. Taking proteomeanalysis as an example, the apparatus and measurement system accordingto the invention are described below. This analysis example is aproteome analysis example concerning a species of organism for whichgenome decipherment has been completed, and it is an example of theso-called shotgun method.

[0036] According to the shotgun method, the molecular weights of partialfragments of proteins are determined by mass spectrometry, and theoriginal proteins are identified by checking a database for amino acidsequences translated from genomic base sequences. Initially, a proteinmixture extraction from cells is decomposed with a digestive enzyme, orthe like, to give a peptide mixture. A sample solution containing theresulting peptide mixture is loaded into the injector of a liquidchromatograph (LC) 60 and injected into the LC flow channel. The peptidemixture in the sample is separated into molecular species according tothe molecular weight during passage through the separation column, andthose species arrive one by one at the electrospray (ESI) ion source 1connected to the LC flow channel terminus in about several minutes toseveral hours after sample injection. The ion source 1 is not limited tothe ESI. The ion source 1 is always in operation, and the peptidefragments that have arrived at the ion source are ionized in order ofarrival.

[0037] The ions formed are introduced into the mass spectrometer throughthe aperture 2, then pass through the gate electrode 4 and enter the iontrap 5 disposed within a first vacuum region 3. 50 and 51 are powersupplies connected to the gate electrode 4. The ion trap 5 is comprisedof a ring electrode 15 and two endcap electrodes 16 and 17. The ringelectrode 15 is connected with a DC power supply 43 and a high-frequency(AC) power supply, and the endcap electrodes 16 and 17 are connectedwith DC power supplies 41, 44 and high frequency (AC) power supplies 42,45, each via a switch 48, respectively. The switching (on-and-off)timing of the switch 48 is controlled by a controller 14. In FIG. 1,there is shown a gas supply pipe 6; in principle, however, this isunnecessary.

[0038] In accumulating ions, a high-frequency voltage is applied to thering electrode 15, while the two endcap electrodes 16, 17 are grounded.By this, a quadrupole electric field is formed within the ion trap 5 andcan entrap those ions not lower in mass-to-charge ratio (m/z) than thatcorresponding to the amplitude of the high-frequency voltage among theincoming ions. After about 1 to 100 ms of ion accumulation in thatmanner, the voltage of the gate electrode 4 is changed (via switch 52)to thereby stop ions from entering the ion trap. In this state, the ionsentrapped are stabilized for about 0 to 10 ms.

[0039] Thereafter, the high-frequency voltage application to the ringelectrode 15 is discontinued and, immediately thereafter, a DC voltageof about 0 to 100 V is applied to the ring electrode 15 and two endcapelectrodes 16, 17 (rise time about 10-100 ns) to thereby form anacceleration electric field within the ion trap 5. The accelerated ionsare discharged from the ion trap 5 and pass through the pinhole 7, whichis grounded. The kinetic energy of an ion in the axial direction of theion trap after passage through the pinhole 7 is determined by thepotential Vtrap in the central part of the ion trap 5 but does notdepend on the mass number of the ion.

[0040] The ion that has passed through the pinhole 7 flies at a velocityv determined by (M/z)·v²=2 eVtrap and passes through the orthogonalaccelerator 18. Here, M is the mass of the ion, z is the valence of theion, and e is the elementary electric charge. Therefore, an ion smallerin m/z arrives at the accelerator 18 earlier.

[0041] The orthogonal accelerator 18 is comprised of two parallel plateelectrodes 9 and 10 and is disposed in a second vacuum region 8. Whilethe orthogonal accelerator 18 is filled with ions, the two electrodes 9,10 are grounded and, after completion of ion filling, a high-voltagepulse is applied to the acceleration electrode 9 (rise time 10 to 100ns). The electrode 10 is in a mesh form for allowing passage of ions,with the periphery being in a plate form, and the outward form thereofis almost equal to that of the electrode 9. Therefore, the ions thathave entered the orthogonal accelerator 18 after application of theacceleration voltage to the acceleration electrode 9 are immediatelyaccelerated and collide against the periphery of the electrode 10 but donot arrive at the detector. The ions that have passed through the meshedportion of the electrode 10 fly through the electric field-free driftspace 11 and enter the reflectron 12 and are inverted within thereflectron and again fly through the drift space and enter the MCPdetector 13. The use of the reflectron 12 is advantageous in that thetime divergence due to the spatial spreading (in the direction ofacceleration) of ions in the orthogonal accelerator 18 can thereby befocused to improve the resolving power and in that the apparatus can bemade smaller. By dividing the orthogonal accelerator 18 into twoacceleration electric field stages and adjusting the space focal planeusing the principle of two-stage acceleration, it is possible tooptimize the focusing effect of the reflectron 12.

[0042] The flying direction of ions that have entered the drift space 11has a certain angle a relative to the direction of the accelerationelectric field. The angle a of ion flight depends upon Vtrap and theinitial voltage Vacc within the orthogonal accelerator 18, but does notdepend on m/z. Therefore, for detecting all ions that are accelerated,the detector used should be at least equivalent in length to theacceleration region. The magnitudes of Vtrap and Vacc are, for example,20 V and 7.5 kV, respectively, and a is about 3 degrees.

[0043] When, in the above case, ion trajectories are focused by using anelectrostatic lens 30, the detector 13 can be made smaller in size. Atthe same time, by disposing the electrostatic lens 30 between the iontrap outlet and the pinhole 7, it is possible to increase the amount ofions passing through the pinhole and to improve the detectionsensitivity. At the same time, the spreading of ion beams can besuppressed, and the resolution can be improved. By switching theswitches 48, 49 and 52, the controller 14 controls the magnitudes of thevoltage to be applied to the gate electrode 4, ring electrode 15, endcapelectrodes 16, 17 and orthogonal accelerator 18 as well as the timingsof application thereof.

[0044] The time from ion ejection from the ion trap 15 to theapplication of a pulse voltage to the orthogonal accelerator 18 iscontrolled by a delay circuit disposed within the controller 14. Therelationship between the delay time and the m/z range of ions to bedetected is determined by the electrode disposition from the ion trap 5to the orthogonal accelerator 18 and by each electrode potential intransferring ions from the ion trap to the orthogonal accelerator 18.Therefore, the delay time is determined in advance according to the m/zrange of ions to be detected. The controller 62 is superior to thecontroller 14 and interlocks the timing of starting measurement by thedetector 13, the operational control of the orthogonal accelerator 18 bythe controller 14, and other similar operations.

[0045]FIG. 2 shows the voltage sequence applied to the respectiveelectrodes in carrying out ordinary MS analysis. After ion ejection fromthe ion trap, the voltage of each electrode in the ion trap is switchedfrom the DC voltage for acceleration electric field formation to avoltage for forming a quadrupole electric field. Immediately thereafter(after about 1 μs), the gate voltage is changed to restart ion injectioninto the ion trap. Thereafter, an acceleration voltage pulse is appliedto the orthogonal accelerator. The pulse width of the accelerationvoltage pulse is set at a level somewhat longer than the time requiredfor all ions occurring in the acceleration region to enter the driftspace. This time depends on the range of mass-to-charge ratios of ionsoccurring in the acceleration region. This mass-to-charge ratio range(hereinafter, “mass window”) depends on the time from just afteracceleration electric field formation in the ion trap to the applicationof the acceleration voltage pulse (Tacc in the figure).

[0046] The mass window is selected by a technician or operator and isinput through the keyboard of a computer. The ratio Mmax/Mmin betweenthe maximum value Mmax and the minimum value Mmin of the mass windowdoes not depend on Vtrap; but rather is constant. Therefore, theoperator need only input Mmin (or Mmax) alone. Alternatively, a systemmay be employed in which a plurality of appropriate mass windows areprepared in advance, for example, on the display of a personal computer,and the operator selects one of these mass windows. The timing ofacceleration pulse application and the acceleration pulse width arepreferably automatically calculated by software.

[0047] Generally, mass spectrometry is repeated about 10 to 1,000 timesto obtain an integrated spectrum. Thereafter, the peak showing thehighest intensity is selected from among the MS spectrum thus obtained,and MS/MS analysis is performed. This selection is preferablyautomatically made by software. In MS/MS analysis, like in the case ofMS analysis, ions are accumulated in the ion trap. Then, ions other thanthe ion corresponding to the selected ion (called the “parent ion”) aredischarged from the ion trap, and the parent ion is decomposed by CID.Some of all of the fragment ions (called “daughter ions”) formed upondecomposition of the parent ion are entrapped and accumulated in the iontrap. Then, the daughter ions are ejected from the ion trap using thesame sequence as that shown in FIG. 2 and subjected to TOFMS analysis.

[0048] Generally, the above sequence is repeated about 10 to 100 timesand the MS/MS spectral data obtained are stored in a recording medium.After completion of analysis of the sample solution, the MS/MS spectraare integrated, and the molecular weight of each daughter ion iscalculated. For the ESI method, which, in particular, tends to allow theformation of multivalent ions, it is first necessary to determine thevalence of each ion. Since a protein contains a large number of carbonatoms, the valence of a fragment ion can be determined based on thedistance between isotope peaks due to stable carbon isotopes. Theaverage molecular weight of each daughter ion is then determined basedupon the isotope peak intensity ratios and the valence. By checking themolecular weight obtained against a database 61 (FIG. 10), the originalprotein is identified.

[0049] A peak showing the second highest intensity is the selected fromamong the MS spectrum and subjected to MS/MS analysis in the samemanner. Thereafter, MS/MS analysis is performed upon successivelydecreasing peaks until the peak with the nth highest intensity isanalyzed. Generally, n is approximately 1 to 5 and is selected inadvance by the measuring personnel. The above series of measurements isrepeated on a mass spectrometer until completion of the analysis of thesample solution.

[0050] Generally, one MS spectrometric measurement and one MS/MSspectrometric measurement require 0.1 to several seconds, respectively,and one series of measurements requires several to scores of seconds intotal. On the other hand, each peptide fragment eluted from an LC isintroduced into the mass spectrometer for scores of seconds to severalminutes. Therefore, the series of measurement is repeated several timesto scores of times for each peptide fragment.

[0051] In FIG. 3, there is shown the construction of a quadrupole iontrap suited for use in the mass spectrometer of the present invention.The ion trap is comprised of four parallel plate electrodes 21 to 24.The two terminal ones are endcap electrodes 21 and 24, and theintermediate two are ring electrodes 22 and 23. For accumulating ions,the same high-frequency voltage, identical in amplitude, frequency andphase, is applied to the two ring electrodes 22 and 23, while the twoendcap electrodes are grounded. For ejecting ions, an appropriate DCvoltage is applied to the four electrodes to thereby form anacceleration electric field. The use of a plane quadrupole ion trapenables the formation of a uniform acceleration electric field and isadvantageous in that: (1) the ion beam spreading is slight; (2) thecontrol of the space focal plane by two-stage acceleration is easy; and(3) the spatial focusing effect is also good. By disposing the spacefocal plane by two-stage acceleration at the detection site or in thevicinity thereof, it becomes possible to reduce the spreading of ionswithin the detection plane and suppress the detection sensitivity fromdecreasing in the terminal portions of the mass-to-charge ratio range.

[0052] Resonance emission is utilized as a means for dischargingunnecessary ions other than the parent ion from the ion trap. Ineffecting resonance emission, an AC voltage with a frequency of f isapplied between a pair of endcap electrodes. On that occasion, thetrajectory of ions having an m/z corresponding to the frequency f israpidly expanded and the ions are discharged from the ion trap. Whenscanning is carried out with this frequency f in a predeterminedfrequency range exclusive of the vicinity of the frequency f0corresponding to the m/z of the parent ion, ions other than the parention are discharged from the ion trap. This resonance emission may alsobe effected simultaneously with the entrapment and accumulation of ionsin the ion trap. In this case, the accumulation of ions and thedischarging of unnecessary ions are carried out simultaneously, suchthat the cycle of repetition of analysis is shortened and, as a result,the sensitivity is improved.

[0053] It is also possible to discharge unnecessary ions by applyingdesired frequency components other than the frequency f0 and thevicinity thereof simultaneously in an overlapping manner, rather than byscanning with the frequency f. When this technique is employed, nofrequency scanning is necessary; hence, the time required fordischarging unnecessary ions may advantageously be curtailed. Othermethods, for example a method comprising applying a DC voltage with ahigh-frequency voltage in an overlapping manner to a ring electrode, canalso be used for eliminating unnecessary ions. This method, however, iscomplicated in voltage control, and the method utilizing resonanceemission is more practical.

[0054] In FIG. 4, an example of the ion trap controlling method by whichthe ion velocity distribution can be reduced is shown. After ionaccumulation in the ion trap, the high frequency voltage application isdiscontinued, and a DC voltage then is applied to two endcap electrodesand a ring electrode to form an accelerating electric field within theion trap. On that occasion, each electrode potential is gradually variedfrom the ground potential level such that the gradient of theaccelerating electric field may be increased. The gradual change inelectrode potential is effected by means of a voltage scanning circuitadapted to the DC power supply. When the maximum voltage value (absolutevalue) and the time required for reaching that maximum voltage value areset up, the voltage scanning circuit can realize arbitrary voltagescanning.

[0055] When ions are ejected by means of a constant acceleratingelectric field, the kinetic energy of ions ejected from the ion trap isconstant. The velocity v of an ion ejected is defined by v={squareroot}{square root over ( )}(2(z/M)eV). Here, M is the mass of the ion,and V is the potential in the central portion of the ion trap. Thus,when the accelerating electric field is increased, the kinetic energy ofan ion ejected increases with the increase in m/z. Therefore, when them/z has a larger value, V in the above velocity formula is also larger.By adequately selecting the increment in accelerating electric field andthe increasing velocity, it is possible to expand the mass-to-chargeratio range that may be analyzed at a single time and, at the same time,reduce the size of the detector.

[0056] In FIG. 5, there are schematically shown ion trajectories for (a)a case where the accelerating electric field is not increased and (b) acase where the acceleration electric field is increased appropriately.The same effect can also be achieved by increasing the acceleratingelectric field stepwise.

[0057]FIG. 6 shows an ion trap controlling method by which theaccelerating electric field is increased stepwise. The method comprisinga stepwise increase in the accelerating electric field is advantageousin that the spatial spreading of ions due to the turnaround time can besuppressed.

[0058]FIG. 7 shows an example of apparatus construction and of thecontrolling method by which the velocity distribution of ions can bereduced. An electrode 65 is disposed between the ion trap 5 andorthogonal accelerator 18. The electrode 65 is generally set at apotential such that a decelerating electric field is formed between itand the ion trap outlet side. The RF voltage application to the ringelectrode 15 is discontinued, and an accelerating electric field isformed within the ion trap 5 to eject the ions accumulated in the iontrap. While ions are ejected and pass through the decelerating electricfield, the potential of the electrode 65 either: (a) decreases thegradient of the decelerating electric field; (b) causes the deceleratingelectric field to disappear; or (c) forms an accelerating electricfield, as shown in the figure. By optimizing the change in deceleratingelectric field and the timing of changing, the same effect as that shownin FIG. 5 can be achieved. The optimizing conditions are formularizedand stored in the software for measurement, and the measuring operatormay only be required to designate the minimum mass (or maximum mass).

[0059]FIG. 8 shows, as an example, the results of calculation concerningthe mass-to-charge ratio range enlarging effect of the above-mentionedmethod. The electrode construction and voltage controlling method are asshown in FIG. 8(a). The ion trap used is of the plate type, and themulti-stage acceleration method is used for optimizing the space focalplane. An electrode is disposed behind the outlet of the multi-stageaccelerator to form a decelerating electric field between themulti-stage accelerator outlet (ground potential) and the electrode, andthe decelerating electric field is caused to disappear at a certaintiming during passage of the ions therethrough by changing the electrodepotential to the ground potential.

[0060] The calculation results shown in FIG. 8(b) are for the case wherethe present method is used, and those shown in FIG. 8(c) are for thecase where the present method is not used, namely the case where theelectrode is always at ground potential. In each graph, the firstordinate axis denotes the position of ions at the time of accelerationpulse application to the orthogonal accelerator. Here, the position 0 mmcorresponds to the accelerator inlet, and the position 50 mm to theaccelerator outlet. From the figures, it is seen that when the presentmethod is used, ions with m/z 500 to 3,100 occur in the accelerationregion at the time point of acceleration pulse application. The ratiobetween maximum mass and minimum mass (Mmax/Mmin) is 6.2. On the otherhand, when this method is not used, ions with m/z 600 to 1,600 occur inthe acceleration region, and the ratio Mmax/Mmin is 2.7. Thus, the masswindow is about 2.3-fold enlarged with the present method.

[0061] In each graph, the second ordinate axis denotes the kineticenergy of ions in the orthogonal accelerator. Using the position andkinetic energy obtained by this calculation as initial conditions, theion trajectories in the TOF segment may be calculated using the iontrajectory analysis software “SIMION,” whereupon it is revealed that thespatial distribution of ions on the detection face of the detector iswithin 13 mm when the present method is used. When this method is notused, the spatial distribution on the detection face is equal to thelength of the acceleration region, as mentioned above, namely 50 mm.Thus, the size of the detector can be reduced to about one third itsconventional size.

[0062] As an alternative to this method, a method comprising changingthe potential of the endcap electrode on the outlet side of the ion trapduring passage of ions between the endcap on the outlet side and theelectrode may be used to produce the same effect. Alternatively, thepotentials of both the outlet side endcap and the electrode may bechanged. In summary, the only requirement is to change the electricfield between both the electrodes such that the ratio in kinetic energybetween preceding ions and succeeding ions among the ions flying betweenboth the electrodes can be reduced. For reducing the dispersion of theion beam, however, the method comprising decelerating preceding ions ispreferred to the method comprising accelerating succeeding ions.

[0063] This method is also effective in an orthogonal acceleration typeTOFMS in which a linear trap (two-dimensional ion trap) is used. Themeans for reducing the velocity distribution of ions may also utilize amagnetic field, rather than an electric field.

[0064] As the means for ejecting ions from the ion trap, the methodwhich comprises discontinuing RF voltage application for ionaccumulation and then forming an accelerating electric field within theion trap is preferably used. When an accelerating electric field isformed while applying an RF voltage, the spatial distribution of ionswithin the ion trap, the kinetic energy distribution for the ions withinthe ion trap, and the spatial dispersion of ions in the accelerationregion due to impact scattering by collision with neutral gas moleculesincreases. When the present method is used, no such increasing effectsare produced.

[0065] Ions within the ion trap show spatial distribution to a certainextent, such that even when the above-mentioned ion ejecting means isprovided, the ions differ in initial potential at the time of ejectionowing to their differing initial positions. Ions on the remote side fromthe outlet are ejected later than the ions on the close side to theoutlet. Because, however, the velocity of the former ions is higher ascompared with the ions on the close side to the outlet, the formerovertake the latter at a certain position. This position is called the“space focal plane”. By forming an electric field for accelerating ionsin the direction of movement thereof between the ion trap outlet to theorthogonal accelerator, it is possible to adjust the position of thespace focal plane according to the well-known principle of multi-stageacceleration. By optimizing the position of the space focal planeaccording to this principle, it becomes possible to improve theefficiency of detection of ions occurring in the acceleration regionterminus.

[0066] Second Exemplary Embodiment

[0067]FIG. 9 shows an example of the analytical sequence using thesegment method according to the present invention. In the segmentmethod, a mass-to-charge ratio range to be analyzed is divided intoseveral segments. In the example shown here, an m/z range of 200 to3,200 is analyzed using an apparatus with Mmax/Mmin=2. In this case, thewhole mass-to-charge ratio range is divided into 200 to 400 (mass window1), 400 to 800 (mass window 2), 800 to 1,600 (mass window 3) and 1,600to 3,200 (mass window 4). Considering the sensitivity decrease at theend portions of each mass window, the respective neighboring masswindows are terminally overlapped to an appropriate extent. In joiningthe mass spectra together, the spectrum higher in intensity is selectedout of the two spectra of the respective windows in each overlappingmass range.

[0068] Initially, ions are accumulated in the ion trap, the ions arethen ejected from the ion trap, and an acceleration pulse is applied foranalyzing the mass window 1. A second acceleration pulse is then appliedfor analyzing the mass window 3. Thereafter, ions are accumulated again,and mass windows 2 and 4 are analyzed in the same manner. When thenumber of mass windows is larger, the whole range can be analyzed by twoperiods of ion accumulation while increasing the number of accelerationpulses to be applied following each time of ion accumulation. Themeasuring person is required only to select the mass-to-charge ratiorange to be analyzed. The mass window setting and the timing of eachacceleration pulse application are automatically determined orcalculated by the appropriate software.

[0069] Since the possibility of daughter ion peaks overlapping with theparent ion peak is low, the necessity of analyzing the region close tothe parent ion peak is not great. In the ion trap, daughter ions havingnot higher than ⅓ or not lower than 3 in m/z ratio to the parent ion arenot accumulated. Therefore, when an apparatus withMmax/Mmin=approximately 3 is used, it is sufficient to analyze tworegions lower and higher than the parent ion peak, excluding thevicinity of that peak following one ion accumulation process.

[0070] Third Exemplary Embodiment

[0071]FIG. 10 shows a hybrid apparatus according to the inventioncomprised of an ion trap type mass spectrometer and an iontrap-connected time-of-flight mass spectrometer of the orthogonalacceleration type. This apparatus is constructed by disposing a detector68 for detecting ions deflected by deflection electrodes 66 and 67 inthe ion trap-connected time-of-flight mass spectrometer of theorthogonal acceleration type. In ion trap mass spectrometry, a massspectrum is obtained by scanning with a high frequency voltage amplitudeto discharge ions from the ion trap in an increasing order of m/z, anddetecting the same. In this hybrid apparatus, a potential difference isgiven between the two deflection electrodes and scanning is made with ahigh frequency voltage, and the ions discharged are deflected anddirected to the detector. Out of the two deflection electrodes, the onethrough which ions pass is in a mesh-like form. It is also possible todeflect ions by providing a potential difference between the otherelectrode and the plane of incidence of the detector in lieu of the useof the mesh-like electrode. This detector may also be disposed behindthe orthogonal accelerator. In this case, the deflection electrodes 66,67 are no longer necessary, and the apparatus construction issimplified. However, the sensitivity is sacrificed due to the occurrenceof a pinhole in the middle of the route of ions.

[0072] The amplitude of the high frequency voltage is then fixed at anappropriate value, and the ions remaining in the ion trap are stabilizedfor about 0 to 10 ms, during which the function of the deflectionelectrodes is ceased. Thereafter, TOFMS analysis is performed. Even withan apparatus with Mmax/Mmin=approximately 2, this method makes itpossible to analyze an m/z range as wide as 100 to 3,000 by one ionaccumulation procedure by, for example, analyzing the m/z range of 100to 1,500 by ion trap mass spectrometry and analyzing the m/z range of1,500 to 3,000 by the TOFMS. This method may be combined with the methodof enlarging the mass windows by reducing the velocity distribution ofions and, by this combination, a broader mass-to-charge ratio range canbe measured with high resolution.

[0073] In proteome analysis using the shotgun method, a higher level ofmass resolution is more advantageous in determining the valences ofdaughter ions. When, however, the parent ion is selected, suchresolution power as for daughter ions is not necessary, but rather, thedetection sensitivity is more important. Generally, MS/MS measurementscan attain higher sensitivity as compared with MS measurements. Thereasons for this include: in MS/MS measurements, ion accumulationconditions can be selected solely for the target parent ion; that otherions and chemical noises can be markedly reduced in the process ofisolation; and that decomposition of the parent ion to lower molecularweight compounds results in a decrease in the number of isotope peaksand an increase in peak intensity per peak. When the ITMS and orthogonalacceleration type IT-TOFMS are compared, the ITMS is higher insensitivity in some cases according to the measurement conditions andapparatus constitution. When this hybrid apparatus is used, it ispossible to use the ITMS for MS spectrum measurements and the TOFMS forMS/MS spectrum measurements. The parent ion selection efficiency isthereby improved and, as a result, the protein identification efficiencyis improved.

[0074] Fourth Exemplary Embodiment

[0075] In FIG. 11, another example is shown of the construction of amass spectrometer according to the present invention. Ions formed in theion source are introduced into a quadrupole ion trap disposed in a firstvacuum region 3 within a vacuum system. The ions are trapped andaccumulated in the ion trap for a certain period of time and thenejected from the ion trap. The ions ejected pass through a pinhole 7 andenter a second vacuum region 8 in which a time-of-flight measuringdevice is disposed. An orthogonal accelerator is disposed in the secondvacuum region 8 and can form an electric field for accelerating the ionsafter passage through the pinhole 7 in the direction orthogonal to theaxial direction of the ion trap (direction of ejection of ions).Initially, no electric field is formed in the orthogonal acceleratorand, while the ions to be detected are passing through the orthogonalaccelerator, a pulse voltage is applied to form an accelerating electricfield.

[0076] Based on the time of flight of an accelerated ion until arrivalat the detector 13, the ratio m/z of the ion can be determined. Since aninert gas (e.g., helium or argon) has been introduced into the ion trapinside for the purpose of increasing the trapping efficiency, the degreeof vacuum within the ion trap is about 1 mTorr, and the degree of vacuumoutside the ion trap but within the first vacuum region 3 is about 10μTorr. The first vacuum region 3 and second vacuum region 8 areseparated from each other by a partition wall having only a pinhole 7with a diameter of about 1 to 2 mm, and are under high vacuum (about 0.1μTorr). Since the accelerator is disposed in such a high vacuum regionof about 0.1 μTorr, ions rarely collide with neutral gas moleculesduring acceleration or after acceleration until arrival at the detector.A high level of resolution can thus be realized.

[0077] The ions ejected from the ion traps arrive at the orthogonalaccelerator in an increasing order of m/z thereof, such that only thoseions passing through the accelerator at the time of pulse voltageapplication to the orthogonal accelerator are detected. However, in thepresent apparatus, ions can be focused, by using a quadrupole ion trap,in a very narrow region (for example, not more than about 1 mm indiameter) in the central portion of the ion trap, so that the spatialdistribution of ions having the same m/z in the axial direction in theorthogonal accelerator is narrow; the apparatus is thus characterized inthat the detection sensitivity thereof is high as to ions to bedetected.

[0078] Fifth Exemplary Embodiment

[0079] While in the first exemplary embodiment the ion velocitydistribution is narrowed by switching the voltage polarity applied tothe ring electrode and endcap electrodes disposed in the ion trap fromalternating to direct, the same effect can be produced by disposing themeans for reducing the ion velocity distribution outside the ion trap.Thus, the ion velocity distribution reducing effect can be produced bydisposing, outside the ion trap, parallel electrodes connected to a DCcurrent power supply and applying a DC voltage to ions ejected from theion trap.

[0080] By enlarging the mass-to-charge ratio range analyzable per ionaccumulation in an ion trap-connected time-of-flight mass spectrometerof the orthogonal acceleration type as an MSn apparatus with highresolution and high sensitivity, the practicability thereof in proteomeanalysis is improved and, as a result, the efficiency of proteinidentification is improved.

[0081] Nothing in the above description is meant to limit the presentinvention to any specific materials, geometry, or orientation of parts.Many part/orientation substitutions are contemplated within the scope ofthe present invention. The embodiments described herein were presentedby way of example only and should not be used to limit the scope of theinvention.

[0082] Although the invention has been described in terms of particularembodiments in an application, one of ordinary skill in the art, inlight of the teachings herein, can generate additional embodiments andmodifications without departing from the spirit of, or exceeding thescope of, the claimed invention. Accordingly, it is understood that thedrawings and the descriptions herein are proffered by way of exampleonly to facilitate comprehension of the invention and should not beconstrued to limit the scope thereof.

What is claimed is:
 1. A mass spectrometer, comprising: an ion source;an ion trap for accumulating the ions formed in said ion source andejecting the same; means for reducing the velocity distribution of theions ejected from said ion trap; a first voltage applying means forapplying a voltage, in a transverse direction relative to the directionof ion ejection, to the ions ejected from said velocity reducing means;and a detector for detecting the ions to which the voltage has beenapplied in the transverse direction.
 2. A mass spectrometer according toclaim 1, wherein said velocity reducing means comprises a second voltageapplying means for applying a voltage to the ions ejected from said iontrap.
 3. A mass spectrometer according to claim 1, further comprising: aring electrode and endcap electrodes in said ion trap; a first directcurrent (DC) power supply and first alternating current (AC) powersupply for supplying electric power to said ring electrode; a second DCpower supply and second AC power supply for supplying electric power tosaid endcap electrodes; and switching means for switching between thefirst DC power supply and first AC power supply and between the secondDC power supply and second AC power supply, respectively.
 4. A massspectrometer according to claim 3, wherein the first DC power supply orsecond DC power supply is equipped with a voltage scan circuit for thestepwise application of DC voltages.
 5. A mass spectrometer according toclaim 3, wherein the first DC power supply or second DC power supply isequipped with a voltage scan circuit for a ramped application of DCvoltages.
 6. A mass spectrometer as claimed in claim 1 which furthercomprises an electrostatic lens disposed between said first voltageapplying means and said detector.
 7. A mass spectrometer, comprising: anion source; an ion trap for trapping the ions formed in said ion source;means for discharging part of the ions trapped from said ion trap inorder of increasing mass-to-charge ratio; a first detector for detectingthe discharged ions; means for ejecting the ions trapped by said iontrap; means for applying a voltage, in the transverse direction relativeto the direction of ion ejection, to the ions ejected from said iontrap; and a second detector for detecting the ions to which the voltagehas been applied in the transverse direction.
 8. A mass spectrometeraccording to claim 7, further comprising: means for deflecting thetrajectory of ions discharged from the ion trap into said firstdetector.
 9. A mass spectrometer, comprising: an ion source; an ion trapfor accumulating the ions formed in said ion source and ejecting thesame; means for controlling the timing of ion ejection from said iontrap; a first voltage applying means for applying a voltage, in atransverse direction relative to the direction of ion ejection, to theions ejected from said ion trap; a controller for interlocking saidfirst voltage applying means with said means for controlling the timingof ion ejection, said controller determining the period between thetiming of starting ion ejection and the timing of starting the operationof said voltage applying means, according to the range of mass-to-chargeratios of the ions to be identified; and a detector for detecting theions to which the voltage has been applied in the transverse direction.10. A mass spectrometer according to claim 9, wherein said controllervaries the period between the timing of starting ion ejection and thetiming of starting the operation of the voltage applying means in a waysuch that multiple mass-to-charge ratio ranges can be analyzed.
 11. Amass spectrometer according to claim 9, wherein said controller causessaid first voltage applying means to apply the transverse voltage aplurality of times from the initiation of ion ejection so that multiplemass-to-charge ratio ranges can be analyzed.
 12. A mass spectrometeraccording to claim 11, wherein the ion ejection and application of aplurality of transverse voltages are repeated and the timing ofapplication of said plurality of transverse voltages differs per eachrepeated ion ejection.
 13. A mass spectrometer according to claim 11,wherein said controller determines the period between the timing ofstarting ion ejection and the timing of starting the operation of thevoltage applying means such that each mass-to-charge ratio region forion detection may partly overlap with the preceding one and/orsucceeding one per application of the transverse voltage.
 14. A massspectrometer according to claim 1, wherein said ion trap is a quadrupoleion trap.
 15. A mass spectrometer according to claim 1, furthercomprising: means for selecting a group of ions among the ions trappedin said ion trap; means for discharging, from the ion trap, ions otherthan the selected ions while retaining said selected ions within the iontrap; and means for dissociating the selected ions within the ion trap.16. A mass spectrometer according to claim 15, wherein said means fordischarging comprises a pair of electrodes and an alternating current(AC) power supply for applying an AC voltage between said electrodes andscanning a frequency within a selected frequency range.
 17. A massspectrometer according to claim 16, wherein said means for dischargingapplies a voltage containing frequency components other than saidselected frequency range between said pair of electrodes.
 18. A massspectrometer according to claim 1, wherein said means for ejecting ionsfrom the ion trap in a predetermined direction comprises means forapplying an alternating current (AC) voltage and a direct current (DC)voltage to said ion trap, and a controller for controlling the order ofapplying said AC voltage and DC voltage, said controller allowing ACvoltage application and, after termination of the AC voltageApplication, allowing DC voltage application.
 19. A measurement system,comprising: a liquid chromatography; and a mass spectrometer comprisingan ion source, an ion trap for accumulating the ions formed in said ionsource and ejecting the same, means for reducing the velocitydistribution of the ions ejected from said ion trap, a first voltageapplying means for applying a voltage, in a transverse directionrelative to the direction of ion ejection, to the ions ejected from saidvelocity reducing means, and a detector for detecting the ions to whichthe voltage has been applied in the transverse direction.
 20. Ameasurement system according to claim 19, further comprising: a databaseholding information pertaining to proteome analysis.